Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency

Abstract

Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient's leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient's dermal fibroblasts and induced pluripotent stem cell (iPSC)-derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.

Fig. 1. Autosomal recessive IRF7 deficiency from compound heterozygous mutations

(A) Familial segregation of IRF7 mutations in a nonconsanguineous French family. (B) Schematic illustration of IRF7A featuring DNA binding domain (DBD), constitutive activation domain (CAD), virus-activated domain (VAD), inhibitory domain (ID), and signal response domain (SRD). A potential nuclear localization signal (NLS) lies between amino acids 417 and 440, and the nuclear export signal (NES) between amino acids 448 and 462. Phosphorylation sites (P) Ser⁴⁷⁷ and Ser⁴⁷⁹ lie at the C terminus. Mutations are shown in red. (C) Wild-type, F410V, or Q421X IRF7 activation of IFNB, IFNA4, or IFNA6 promoter-driven reporter assay. Cells are uninfected (UI) or infected with SeV. Means ± SD of three independent experiments are shown. *P < 0.01, **P < 0.005, ***P < 0.001 as determined by t test. (D) Phosphorylation of HA-tagged wild-type (WT), F410V, or Q421X IRF7 coexpressed with FLAG-tagged TBK1 as assessed by Western blot with phospho-specific IRF7 antibody (P-IRF7); GAPDH was used as a loading control. This result is representative of two experiments.

Fig. 2. P’s IRF7 alleles are loss-of-function by different mechanisms

(A to C) Localization of FLAG-tagged wild-type (A), F410V (B), or Q421X (C) IRF7 in uninfected or Sendai virus–infected Vero cells by immunofluorescence imaging. This result is representative of two experiments. (D) Wild-type and mutant IRF7 dimerization by immunoprecipitation (IP) with antibody to FLAG followed by Western blot with antibodies to FLAG and HA.WCL, whole-cell lysate. (E) Localization of FLAG F410V (top) and FLAG wild-type IRF7 (bottom) cotransfected with HA Q421X IRF7 in Vero cells as assessed by immunofluorescence imaging. This result is representative of two experiments.

Fig. 3. Impaired IRF7-dependent innate immunity in leukocytes and pDCs

(A) The top 5% of genes, in PBMCs infected with pH1N1 at MOI (multiplicity of infection) = 2, whose relative changes were >2 (up or down) in controls and ≤1 in the patient, P, as analyzed by gene array. (B) Causal network analysis of the differentially regulated genes. (C) IFN-α production at 24 hpi with pH1N1 IAV or HSV-1 in pDCs from four healthy controls, P, and an UNC-93B–deficient individual (UNC-93B^−/−), a control for TLR responses. (D) MX1 and IL8 were measured in cells from (C) by qPCR. (E and F) The expression of all indicated type I IFN genes was measured by qPCR in purified pDCs (E) and unsorted PBMCs (F) separately infected with pH1N1 IAV at MOI = 1. The probe for IFNA13 also detects IFNA21 mRNA. All data shown in (C) to (F) are representative of two independent experiments.

Fig. 4. IRF7-dependent intrinsic immunity is required for control of IAV infection

(A) IRF7 mRNA induction at indicated time points after IFN-β treatment in F-SV40. Means ± SD of three replicates are shown. (B) IRF7 expression in F-SV40 measured byWestern blot 18 hours after treatment with IFN-β. (C) Virus titers in F-SV40 from P stably transfected luciferase (Luc) or wild-type IRF7 with an internal ribosome entry site–expressed red fluorescent protein, after infection with pH1N1 (MOI = 10) or VSV (MOI = 3). Means ± SD for pH1N1 IAV (n = 3) and VSV (n = 7) are shown. ***P < 0.001 between controls and P as determined by t test. (D) IRF7 expression in IFN-β– or IFN-λ–treated PECs derived from healthy control ESCs (RUES2), SV iPSCs, or three individual clones (clones 1, 2, and 3) of P’s iPSCs as detected by Western blot. (E) IFN-β production in PECs infected with A/PR/8/34-GFP as measured by ELISA at indicated time points. Means ± SD of two independent experiments are shown. *P < 0.05 as determined by t test. (F) Staining of IAV nucleoprotein (NP) (green) and Nkx2.1 (red) in PECs derived from SV-iPSC control and P infected at MOI = 1 with pH1N1 IAV. Cells derived from a single representative clone of P’s iPSCs are shown. (G) Percentage of Nkx2.1⁺ cells scored as positive for IAV NP, in PECs untreated or treated with IFN-α (100 or 1000 U/ml) for 18 hours and infected with pH1N1 for 24 hours. Means ± SD of two independent experiments are shown. *P < 1 × 10⁻⁶ as determined by χ² analysis.